Physiological and molecular characterisation of circadian rhythms in red blood cells

Lead Research Organisation: University of Surrey
Department Name: Mechanical Engineering Sciences

Abstract

Circadian rhythms allow organisms to actively anticipate, as opposed to passively reacting to, the changes of the 24-hour cycle of a day and a night. These rhythms are generated within our cells, and until recently, the consensus was that they were generated by specific clock genes. The finding, reported three years ago by two of the applicants, that circadian rhythms are also found in isolated human red blood cells (RBCs) was therefore a very remarkable one, because RBCs lack a cell nucleus and therefore do not have any genes. In that report, the protein peroxiredoxin (PRX) was found to alternate rhythmically between an oxidised and a reduced state. Two other applicants have recently found a similar oscillation in the electrophysiological properties of the cell membrane and the cytoplasm that it encloses, using a method called dielectrophoresis (DEP). These two groups have now joined together with the ambition of gaining a greater understanding of how these oscillations are created and connected.

We will sequentially incubate human RBCs in the presence of chemicals that specifically inhibit different groups of biologically active proteins: Protein kinases, which add phosphate groups to other proteins, protein phosphatases, which remove them again, and ion channels, which control the flow of ions across cell membranes and therefore regulate the electrochemical properties of the membrane. Every cell has multiple members of each group, but RBCs, being very specialised cells, have an unusually limited repertoire, and there are chemicals available that act on multiple members of a group, and others that act specifically on a single type. In each case, we will investigate the effect of each chemical both on PRX status and on electrochemical properties. We will interpret a significant effect on the oscillation on both readouts as an indication of a joint mechanism, whereas if it only affects one of them, that protein at least will only affect one of them. This will allow us to identify which proteins within these groups in the RBCs are involved in circadian rhythmicity. We will then proceed to a method known as patch-clamping, which allows us to study a single ion channel molecule within the cell in the presence or absence of an inhibiting chemical.

The outcomes of this research will help us understand how RBCs keep time. These mechanisms may or may not be applicable to other cells. However, RBCs are crucially important for survival, and a greater understanding of how they function may be of benefit to haematology and potentially improved understanding and treatment of malaria, a disease with a strong rhythmic component.

Technical Summary

Multiple biological processes display circadian rhythms controlled by endogenous mechanisms. Current models suggest that this process is driven by the rhythmic expression of clock genes. The recent report that circadian rhythms in redox status are present in purified human red blood cells (RBCs) challenges this paradigm, asking important mechanistic questions. We used dielectrophoresis (DEP) to investigate the electrical parameters of RBCs using human blood samples, and found near-24 h cyclic activity in ionic conductance across the membrane, and an antiphasic rhythmicity in cytoplasmic ionic content. We hypothesised that RBC rhythms would be sensitive to the generic kinase inhibitor, staurosporine; investigation showed an increase in the circadian period of the electrophysiological oscillation caused by this drug, indicating that kinases play a role in this non-transcriptional clock. This proposal is based on the hypothesis that the post-translational rhythms observed in RBCs are modulated by protein phosphorylation and dephosphorylation. We aim to establish which kinase/s and/or phosphatase/s underlie and sustain the circadian clock, which ion channel(s) facilitate(s) the circadian rhythm observed by DEP, and whether electrophysiological rhythms are required for redox oscillations in isolated human RBCs, and vice versa. Ion channels, kinases, and phosphatases in RBCs are limited in number. We will examine the effect of various drugs in order to identify those that affect rhythmic behaviours in DEP and/or redox status. This will provide insight into coupling mechanisms between membrane electrophysiology and RBC metabolism. Inhibitor effects will be investigated sequentially through the same iterative process. Generic inhibitors will be followed by others specific to isoforms. Further investigation using patch clamping (for channel candidates) or other agents such as activators once a change (is observed will examine the direct or indirect effects on ion channels.

Planned Impact

The proposed project will provide impact benefiting not only the academic sector, but also to the public sector, commercial private sector, third sector and the wider public. The major envisioned areas of impact are the following:

Shedding new light on a basic biological mechanism

The initial report of gene-independent circadian rhythms in RBCs by the Cambridge co-applicants received considerable attention both in the scientific community and in news media. We anticipate that, with the proposed continuation of the investigation, there will be a demand for continued educational activities explaining our findings and their implications. The investigators will be working with the press offices of the universities to enable them to publicise our activities and new findings through classical and new media channels, as well as public fora such as science festivals. Recent examples of such participation includes a Brazilian Globo Reporter documentary, viewed by millions, and the Shuffle Festival (London). Future engagements will be tailored according to the progress of the project and suitable opportunities arising. This will include a bid for participation in the Royal Society Exhibition.

Time scale: First public presentations within 12 months.

Dielectrophoresis of RBCs or whole blood as a circadian phase marker

The standard marker of circadian phase is melatonin, which may be sampled and measured from blood, saliva, or (indirectly through its excretion product 6-sulphatoxymelatonin) from urine. Melatonin assays are a very precise and important tool, but have the drawback of not being able to provide an instant answer, as well as the use of a radioimmunoassay involving radioactive iodine. We envision a potential use of dielectrophoresis as a marker of circadian phase that will be faster, cheaper, and safer, and (although not possible with saliva or urine) require a much smaller blood volume.

We can envision the possibility of developing this method commercially with whole blood rather than purified RBCs, which would add greatly to its utility. If the results from the initial phase of our investigation are encouraging for such a development, we will seek development funding and consider partnering with a commercial organisation. Beneficiaries of such as development would involve both basic researchers in human and animal chronobiology and clinicians needing to determine circadian phase for a broad number of different diagnoses.

Timeline: Initiation of development phase after 12 months.

A greater understanding of RBC biology: Implications for haematology research, development, and ultimately clinical practise

The exploration of hitherto unknown and unexpected rhythmic properties of RBC will have important potential impact. Whilst the research proposed in this application is entirely basic in nature, its successful completion will open a novel avenue of investigation. Clinical research settings will be required to establish whether the same rhythms are present in vivo. Whilst that work will not have a major impact on basic research, any RBC rhythms detected cannot be disentangled from entraining cellular and humoral signals, the presence of such rhythms will be of importance to researchers, developers, and ultimately clinicians within the haematology field, in particular with respect to a greater understanding and potentially new treatment avenues for malaria, which is strongly rhythmic in its manifestations.

Timeline: After completion of project

Publications

10 25 50
 
Description We have discovered that red blood cells (RBCs) track the passage of time across a 24-hour period by cyclically altering the level of potassium in the cytoplasm, and that by chemically altering the level of potassium in the cytoplasm, it is possible to alter the period of the cyclic potassium level, or to abolish it entirely.

We have also discovered the role of casein kinase (CK) 1 in temperature compensation in human red blood cells. Specific inhibition of CK 1 resulted in abolishing temperature compensation of RBCs. We show that CK1 activity is essential for circadian rhythms in RBCs regardless of presence/absence of nuclei (or gene expression). This work has just been accepted for publication in Journal of Biological Rhythms. The DOI has yet to be issued for this publication. This will be included in the 2019/20 review period for this award.
Exploitation Route There are a number of significant implications of this finding. Firstly, it demonstrates a non-transcriptional timekeeping mechanism in non-nucleated cells. Secondly, it suggests a new aspect of cellular behaviour with potential implications for multiple aspects of the cell biology of blood, including an understanding of the circadian behaviour of malaria, and a potential new insight into the chronobiological nature of blood-based conditions such as heart attacks and strokes.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Discussions with the manufacturer of the instrument used in our study (the 3DEP from DEPtech, a UK company) suggest that the work has had a positive impact on instrument sales.
First Year Of Impact 2017
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Bank Blood Better: Improving Blood Storage in Hospitals
Amount £87,691 (GBP)
Organisation United Kingdom Research and Innovation 
Sector Public
Country United Kingdom
Start 01/2021 
End 04/2021
 
Description Determining the microbiological safety of Pplus Agitator
Amount £30,000 (GBP)
Organisation University of Southampton 
Sector Academic/University
Country United Kingdom
Start 01/2019 
End 07/2019
 
Description IRG - UK / South Africa Researcher Links Grants for Travel and Hosting of Scientific Events
Amount R33,000 (ZAR)
Funding ID 98108 
Organisation South African National Research Foundation (NRF) 
Sector Public
Country South Africa
Start 06/2015 
End 03/2016
 
Description Investigating the optimum conditions for human platelet isolation and storage
Amount £22,777 (GBP)
Organisation University of Surrey 
Sector Academic/University
Country United Kingdom
Start 08/2018 
End 06/2019
 
Description PhD studentship
Amount £70,000 (GBP)
Organisation University of Surrey 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 10/2020
 
Title Rapid blood cell electrophysiology assay using dielectrophoresis 
Description Following on from our findings that there is circadian variation in the electrophysiology of red blood cells, we have shown that this electrophysiological fingerprint can be determined in under 60 seconds of being drawn from the donor, an has potential in acting as a phase marker for electrophysiological rhythms - with implications for a range of clinical benefits including the timing of cancer therapies, and for the understanding of blood diseases such as malaria. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact We have not yet published this method, though the equipment required to perform the test is available commercially. Not impacts have resulted yet, but we are developing this assay and have sought further funding. 
 
Description Collaboration with the University of York 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution The work has raised awareness of the technology developed and its various applications. Of particular note, its non-invasiveness, label-free and very fast data acquisition. We are currently working at generating preliminary data for a subsequent grant application to Cancer Research UK (CRUK). CRUK had responded positively to our initial application and we have been invited to put a full grant at the next stage.
Collaborator Contribution The partner is providing cells and related media and consumables for data collection. This would provide the necessary preliminary data for our subsequent full grant application to CRUK.
Impact The collaboration is multi-disciplinary and will result in a publication (that is still in preparation) to demonstrate the physical background in obtaining membrane potential using dielectrophoresis. Usually this parameter is obtained by patch clamping or membrane potential sensitive dyes (which can be substrates hence provide artefactual results). The data will also be used to apply for a full grant from CRUK.
Start Year 2015
 
Description Is malarial infection affected by circadian electrophysiology? 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution We travelled to Edinburgh to perform preliminary experiments and have shown circadian effects in malarial infection and related anaemia induced malarial infection.
Collaborator Contribution The team at Edinburgh provided the facilities, expertise, consumables and resources to perform the experiments.
Impact We are currently finalising the data analysis from the preliminary experiments with the aim of working as a multidisciplinary group to apply for further funds from funding bodies such as BBSRC, EPSRC and charities.
Start Year 2017
 
Description Rhythms in platelets 
Organisation University of Reading
Department School of Biological Sciences Reading
Country United Kingdom 
Sector Academic/University 
PI Contribution The award has helped build a new collaboration with the University of Reading to work on platelets.
Collaborator Contribution Prof. Jonathan Gibbins has contributed his extensive platelet expertise to optimise the culture/storage of platelets with minimal acivation, as well as further optimising conditions for further drug treatments.
Impact The work has generated exciting data that is about to be submitted for publication.
Start Year 2019
 
Description A presentation to students and parents with a demonstration-An electrophysiological fingerprint of blood 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Students and parents attended a presentation to demonstrate how an electrophysiological fingerprint from a drop of blood (via a finger-prick) can be obtained. The interactive question and answer session meant that the attendees were involved and they were shown a profile of the electrical properties of blood.
Year(s) Of Engagement Activity 2017
 
Description AES conference (San Francisco, US) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The AES Annual Meeting is the most significant dielectrophoresis meeting worldwide. As meeting organiser in 2016, I set the theme of the conference as "Electrokinetics and Cell Biology". This brought together a wide range of international experts in the field, and allowed a broad dissemination of the work of the group.
Year(s) Of Engagement Activity 2016
 
Description AES conference Salt Lake City 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The session sparked questions and discussion around problems encountered in the research area. The outcomes included troubleshooting, workshops and awareness.
Year(s) Of Engagement Activity 2015
 
Description Clock Club 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The presentation sparked interesting questions and exciting new ideas as feedback.
Year(s) Of Engagement Activity 2015
 
Description Invited AES 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An invited presentation that sparked a number of questions and interest in the field.
Year(s) Of Engagement Activity 2015
 
Description Keynote Speech, Association of Brazilian Postgraduate Students and Researchers in the UK, 11/3/2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I outlined my research and shared my experience as a visiting researcher in Brazil and some career advice for the participants, and afterwards participated in a panel discussion with other keynote speakers.
Year(s) Of Engagement Activity 2018
URL https://sites.google.com/view/xabepukconference/speakers
 
Description Laboratory research experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact As part of promoting STEM related subjects, a student came to the PI lab to gain an insight into the use of dielectrophoresis in a health related area. The student was very keen to learn more about blood and dielectrophoresis having read our recent publication (Henslee et al, Nat Comm, 2017).
The student was particularly interested in shadowing some of the experiments carried out. The student is considering doing Medical Engineering or Medicine.
Year(s) Of Engagement Activity 2018
 
Description National Institutes of Health (NIH) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk to broad audience at NIH in Rockville, MD on electrophysiology in human cells, including red blood cells. The talk generated a lot of interest, questions and debate.
Year(s) Of Engagement Activity 2017
 
Description Potassium regulates electrophysiological rhythms in human red blood cells 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This is the press release for the Nature Communications paper which showed that potassium regulates the human electrophysiological rhythms in red blood cells.
Year(s) Of Engagement Activity 2017
URL https://clinicalnews.org/2017/12/12/potassium-is-critical-to-circadian-rhythms-in-human-red-blood-ce...
 
Description Presentation to Guildford Cardiac Support Group 8/3/2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I gave a presentation entitled "Circadian rhythms, sleep, and cardiovascular health" to the Guildford Cardiac Support Group, an organisation for cardiac patients and their carers. The presentation was attended by approximately 30 individuals, and was followed by a lively questions and discussion section.
Year(s) Of Engagement Activity 2016
 
Description Presentation to the University of the Third Age, Guildford, 10/11/2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I gave a presentation entitled to "Circadian rhythms, sleep, and the life and times of man" to an audience of retirees, and answered a multitude of questions afterwards.
Year(s) Of Engagement Activity 2015
 
Description Radio interview (LBC) with Stig Abell 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Following overnight negotiations at the EU Summit in Brussels, I was interviewed about the effect of sleep deprivation on cognitive function and judgement
Year(s) Of Engagement Activity 2016
 
Description Summer internship 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact The work funded by this grant generated interest within the undergraduate (UG) community. It resulted in UG students taking summer internships in my lab to gain research work experience. One of these UG students (Mr. Gollapudi) is now completing a postgraduate course (MSc) at Imperial College, London.
Year(s) Of Engagement Activity 2018
 
Description Twitter 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I have been on Twitter since 2014, and currently have 660 followers. Many of my tweets are about science - both my own, and the work of other scientists. Through this medium, I initiate and participate discussions and debate about science and science policy.
Year(s) Of Engagement Activity 2014,2015,2016
URL http://www.twitter.com